Summary of work: One theory of aging holds that oxidative damage to cellular components, such as proteins, lipids and DNA, accumulates with age, leading to the cellular dysregulation that result in the process of aging experienced by the organism. We are interested in understanding how exogenous and endogenous sources of reactive species produce oxidative damage in DNA, how that damage is processed in human cells, and the effects of unrepaired DNA damage. Reactive oxygen species produce a wide variety of products in DNA. Differences in how these lesions are processed have made the repair of oxidative damage in DNA difficult to understand. In addition to the complex chemistry of the reactions of these reactive species with DNA and the multiple pathways involved in their repair, at least two of these species also act as intracellular messengers affecting the control of cellular processes. We seek to separate these complexities by introducing well defined oxidative lesions in DNA into cells in vivo or by studying the reactions of cell extracts or purified proteins with DNA containing well defined lesions in vitro. Repair of DNA damage induced by photo activated methylene blue by human whole cell extracts. The damage produced in double-stranded DNA by exposure to visible light in the presence of methylene blue consists almost exclusively of the lesion, 8-oxodeoxyguanosine. This lesion is also produced in significant amounts in DNA, along with many other products, by exposure to gamma irradiation or hydrogen peroxide. By examining the repair of the methylene blue-damaged DNA by proteins extracted from human cells, we have identified two pathways of repair. The kinetics of the two pathways are distinctly different, suggesting different mechanisms involved in repair. In addition, the proportion of the lesions processed by each pathway differs markedly in cell lines derived from patients with the genetic diseases, xeroderma pigmentosum-Group A and Cockayne syndrome-Group B, in comparison with a cell line from an unaffected individual. Both xeroderma pigmentosum and Cockayne syndrome cells have characteristic defects in the repair of damage induced by ultraviolet light and in the processing of other types of oxidative damage. We have identified a protein from mitochondria which is able to incise an oligo containing a site specific 8-oxoguanine adduct. Unlike the bacterial enzyme which cleaves both 3' and 5' to the lesion, the mt Fpg incises exclusively on the 3' side of the lesion. We have finalized our chromatography steps and have begun the physical characterization of the enzyme. We have established an in vitro repair incorporation assay for the detection of removal of the important lesion 8-oxodeoxguanosine. The repair is very efficient as compared to the removal of other lesions such as, for example, UV induced DNA damage. There appears to be two pathways for the repair of this type of oxidative DNA damage, one short patch and the other long patch. In repair deficient human mutant cell lines from xeroderma pigmentosum or Cockayne syndrome cells, one of these pathways appears to be deficient.